BU-4803T Antibiotics

Abstract

 The present invention relates to antitumor antibiotics designated as BU-4803T A₁, A₂, B, C₁, C₂ and D. BU-4803T A₁, A₂ and B are produced by fermentation of Microbispora strain AA9966 which has been deposited with the American Type Culture Collection under the accession number ATCC 55327.

Description

BACKGROUND OF THE INVENTION

[0001] The present invention relates to novel antitumor compounds, the production by fermentation process thereof, and the producing microorganism Microbispora strain AA9966.

SUMMARY OF THE INVENTION

[0002] The present invention provides novel antibiotic compounds herein designated as BU-4803T A₁, A₂, B, C₁, C₂ and D having the physico-chemical properties listed hereinafter. These compounds are useful as antitumor agents; additionally, BU-4803T A₁, A₂ and B are active against Gram-positive bacteria.

[0003] Another aspect of the invention provides a process for the production of BU-4803T A₁, A₂ and B which comprises cultivating an antibiotic producing strain of Microbispora sp. in an aqueous medium containing assimilable sources of carbon and nitrogen under aerobic conditions, and recovering said antibiotic from the fermentation broth.

[0004] A further aspect of the present invention provides a biologically pure culture of a BU-4803T producing strain of Microbispora sp. strain AA9966 having the identifying characteristics of ATCC 55327.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure 1 shows an IR spectrum of BU-4803T A₁.

[0006] Figure 2 shows an IR spectrum of BU-4803T A₂.

[0007] Figure 3 shows an IR spectrum of BU-4803T B.

[0008] Figure 4 shows an IR spectrum of BU-4803T C₁.

[0009] Figure 5 shows an IR spectrum of BU-4803T C₂.

[0010] Figure 6 shows an IR spectrum of BU-4803T D.

[0011] Figure 7 shows a proton NMR spectrum of BU-4803T A₁ (400 MHz, CDCl₃).

[0012] Figure 8 shows a proton NMR spectrum of BU-4803T A₂ (400 MHz, CDCl₃).

[0013] Figure 9 shows a proton NMR spectrum of BU-4803T B (400 MHz, CDCl₃).

[0014] Figure 10 shows a proton NMR spectrum of BU-4803T C₁ (400 MHz, CDCl₃).

[0015] Figure 11 shows a proton NMR-spectrum of BU-4803T C₂ (400 MHz, CDCl₃).

[0016] Figure 12 shows a proton NMR spectrum of BU-4803T D (400 MHz, CDCl₃).

DETAILED DESCRIPTION OF THE INVENTION

[0017] The physico-chemical properties of antibiotic BU- 4803T A₁, A₂, B, C₁, C₂ and b are given below. The optical rotations of the compounds have not been determined because of strong UV absorption at 589 nm. Melting points are determined with a Yanaco micro melting point apparatus and are uncorrected. UV and IR spectra are recorded on a Jasco UVIDEC-610C spectrometer and a Jasco IR-810 spectrometer, respectively. The ¹H and ¹³C NMR spectra are recorded on a Jeol JMN-GX 400 spectrometer with CDCl₃ as an internal standard. FAB-MS are run with a Jeol JMS-AX505H spectrometer using 3-nitrobenzyl alcohol as a matrix. Elemental analysis are measured on Perkin-Elmer 240C Elemental Analyzer. The HPLC is determined using a Cosmosil 5C18 AR column (4.6 mm i.d. x 150 mm, Nacalai Tesque, Inc.); CH₃CN-H₂O (43:57) as the mobile phase at a flow rate of 1.2 ml/min.; and UV absorption at 254 nm as the detection means. The TLC is determined on a RP-18 F_254S plate (Merck, Art 15423) using MeOH-H₂O (87.5:12.5) as the solvent system.
BU-4803T A₁
Nature: red powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide, methanol, acetonitrile, acetone, and methylene chloride
   Insoluble in: Hexane and water
UV λ_max nm (E

):
   in MeOH: 244 (222), 278 (267), 433 (92), 509 (58)
   in 0.1 N HCl-MeOH (1:9): 239 (277), 281 (291), 433 (78), 510 (65)
   in 0.1N NaOH-MeOH (1:9): 236 (217), 278 (292), 437 (119), 613 (51), 648 (50)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1620, 1455, 1405, 1120, 1055 (as shown in Figure 1).
FAB-MS m/z:
   Positive: 1764 (M+H+Na)⁺
   Negative: 1741 (M+2H-H)⁻
Elemental Analysis: C 58.20%; H 6.70%; N <0.3%
HPLC Rt (min.): 10.4
TLC Rf: 0.49
¹H-NMR (CDCl₃): 0.79 (3H, t), 0.87 (3H, t), 0.95 (3H, d), 1.00 (1H, m), 1.04 (3H, d), 1.10 (1H, m), 1.15 (3H, d), 1.29 (3H, d), 1.30 (1H, m), 1.33 (3H, d), 1.34 (2H, m), 1.35 (3H, d), 1.41-1.94 (13H), 1.93 (1H, dd), 1.94 (1H), 1.97 (1H, dd), 2.04 (1H, dt), 2.22 (1H, dt), 2.24-2.38 (2H), 2.24 (3H, s), 2.38 (3H, s), 2.39 (1H, dd), 2.43 (1H, dd), 2.44 (1H), 2.47 (1H, d), 2.64 (1H, d), 2.69 (1H, qui), 3.03 (1H, dd), 3.12 (1H, dt), 3.16 (1H, dt), 3.24-3.33 (2H, dt), 3.34 (1H, d), 3.49 (1H), 3.50-3.60 (2H, dt), 3.64 (1H, dt), 3.68 (1H, dd), 3.72 (1H), 3.73-3.76 (2H), 3.74 (1H), 3.76 (1H), 3.79-3.82 (1H), 3.87 (3H, s), 3.91 (1H, s), 3.95 (3H, s), 3.97 (3H, s), 3.98 (1H), 4.01 (2H, dd), 4.06-4.13 (2H), 4.16 (2H, dd), 4.21 (1H), 4.27 (1H, dd), 4.36 (1H), 4.48 (1H, dd), 4.52 (1H, dd), 4.62 (1H, dd), 4.66 (1H, s), 4.68 (1H, s), 4.94 (1H, d), 5.09 (1H, d), 5.35 (1H, d), 5.43 (1H, d), 5.83 (1H, s), 7.38 (1H, s), 9.69 (1H, s), 12.21 (1H, s), 13.95 (1H, s), 14.96 (1H, s) (as shown in Figure 7)
BU-4803T A₂
Nature: red powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide, methanol, acetonitrile, acetone, and methylene chloride
   Insoluble in: Hexane and water
UV λ_max nm (E

):
   in MeOH: 240 (261), 279 (276), 432 (86), 510 (68)
   in 0.1 N HCl-MeOH (1:9): 239 (275), 280 (288), 432 (76), 509 (70)
   in 0.1N NaOH-MeOH (1:9): 235 (214), 278 (279), 436 (113), 615 (54), 647 (54)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1620, 1455, 1405, 1120, 1055 (as shown in Figure 2)
FAB-MS m/z:
   Positive: 1705 (M+Na)⁺
   Negative: 1683 (M+2H-H)⁻
HPLC Rt (min.): 11.1
TLC Rf: 0.43
¹H-NMR (CDCl₃): 0.78 (3H, t), 0.87 (3H, t), 0.95 (3H, d), 0.99 (1H, m), 1.05 (1H, m), 1.14 (3H, d), 1.25-1.34 (5H), 1.29 (3H, d), 1.30 (3H, d), 1.31 (3H, d), 1.44-2.07 (15H), 1.93 (1H), 1.98 (1H), 2.00 (1H), 2.23 (2H), 2.24 (3H, s), 2.26 (1H), 2.39 (1H, dd), 2.43 (1H, dd), 2.44 (1H), 2.47 (1H), 2.63 (1H, d), 2.69 (1H, qui), 3.03 (1H, dd), 3.12 (1H, dt), 3.16 (1H, dt), 3.20-3.30 (3H), 3.36 (1H, d), 3.49 (1H), 3.47-3.55 (2H), 3.68 (1H, dd), 3.72 (1H), 3.74-3.76 (2H), 3.80 (1H), 3.79-3.82 (1H), 3.87 (3H, s), 3.91 (1H, s), 3.93 (1H, s), 3.95 (3H, s), 3.97 (3H, s), 3.99 (1H), 4.02 (1H), 4.02 (1H), 4.06-4.10 (1H), 4.13-4.17 (2H), 4.21 (1H), 4.27 (1H), 4.35 (1H), 4.42 (1H), 4.49 (1H, dd), 4.52 (1H, d), 4.66 (1H), 4.82 (1H), 4.93 (1H, d), 5.34 (1H, d), 5.43 (1H, d), 5.83 (1H, s), 7.38 (1H, s), 9.69 (1H, s), 12.20 (1H, s), 13.94 (1H, s), 14.96 (1H, s) (as shown in Figure 8)
BU-4803T B
Nature: red powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide, methanol, acetonitrile, acetone, methylene chloride
   Insoluble in: Hexane and water
UV λ_max nm (E

):
   in MeOH: 240 (239), 278 (254), 432 (80), 511 (62)
   in 0.1 N HCl-MeOH (1:9): 239 (260), 281 (271), 431 (71), 510 (67)
   in 0.1N NaOH-MeOH (1:9): 235 (211), 278 (272), 436 (111), 614 (51), 647 (51)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1705, 1620, 1460, 1405, 1125, 1060 (as shown in Figure 3)
FAB-MS m/z:
   Positive: 1749 (M+2H+Na)⁺
   Negative: 1725 (M+2H-H)⁻
Elemental Analysis: C 58.45%; H 6.48%; N <0.3%
HPLC Rt (min.): 18.0
TLC Rf: 0.39
¹H-NMR (CDCl₃): 0.77 (3H, t), 0.85 (3H, t), 0.92 (3H, d), 0.93 (3H, d), 0.97 (1H, m), 1.06 (1H, dt), 1.12 (3H, d), 1.27 (3H, d), 1.30 (1H), 1.31 (3H, d), 1.31 (3H, d), 1.32 (2H), 1.45-1.66 (2H), 1.48 (2H), 1.62 (2H), 1.62-1.96 (4H), 1.66 (2H), 1.88 (1H, dt), 1.92 (1H, dd), 1.93 (1H, dd), 1.98 (2H, m), 1.98 (1H, s), 2.18 (2H), 2.20 (2H), 2.22 (3H, s), 2.23 (3H, s), 2.38 (1H, dd), 2.41 (1H, dd), 2.49 (1H), 2.52 (1H), 2.63 (1H, d), 2.67 (1H, qui), 3.02 (1H, dd), 3.10 (1H, dd), 3.13 (1H, dd), 3.24 (2H, dt), 3.37 (1H, d), 3.47-3.56 (2H), 3.54 (1H, s), 3.55 (1H, s), 3.66 (1H, m), 3.73 (1H, m), 3.74 (1H), 3.80 (1H, m), 3.85 (3H, s), 3.93 (3H, s), 3.95 (1H), 3.95 (3H, s), 3.96 (1H, d), 4.00 (1H, d), 4.08-4.23 (3H, m), 4.25 (1H, dt), 4.25 (1H, dt), 4.37 (1H, s), 4.45 (1H, d), 4.51 (1H, d), 4.66 (1H, s), 4.80 (1H, s), 4.92 (1H, d), 4.92 (1H, d), 5.32 (1H, d), 5.40 (1H, d), 5.81 (1H, s), 7.37 (1H, s), 9.69 (1H, s), 12.20 (1H, s), 13.94 (1H, s), 14.96 (1H, s) (as shown in Figure 9)
¹³C-NMR (CDCl₃): 210.6 s, 210.6 s, 203.5 s, 195.5 s, 187.8 s, 184.8 s, 163.6 s, 158.3 s, 156.8 s, 153.2 s, 151.8 s, 150.6 s, 147.7 s, 139.4 s, 138.5 s, 135.4 s, 125.6 s, 124.7 s, 116.3 s, 113.0 s, 112.1 d, 110.5 s, 108.2 s, 107.9 s, 103.2 d, 103.2 d, 98.9 d, 98.9 d, 98.9 d, 98.7 d, 86.6 d, 85.6 s, 85.4 d, 82.7 s, 79.3 s, 78.9 d, 78.8 d, 78.5 s, 77.2 s, 77.2 s, 77.0 d, 76.3 d, 75.4 d, 75.3 d, 75.2 d, 72.7 d, 72.5 d, 70.6 d, 67.8 d, 67.1 d, 67.0 d, 66.7 d, 66.7 d, 65.1 d, 65.0 d, 61.1 q, 60.9 q, 60.8 q, 55.7 d, 44.4 d, 37.0 t, 35.3 t, 34.8 t, 34.3 t, 30.5 t, 30.4 t, 29.5 t, 29.4 t, 27.8 t, 27.8 t, 27.8 t, 25.0 q, 25.0 q, 24.8 t, 24.8 t, 18.1 q, 18.1 q, 18.0 t, 17.9 q, 17.7 q, 16.6 t, 15.1 q, 14.9 q, 14.6 q, 14.6 q
BU-4803T C₁
Nature: purple powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide and methylene chloride
   Slightly soluble in: Methanol and acetonitrile
   Insoluble in: Acetone, hexane and water
UV λ_max nm (E

):
   in MeOH: 260 (237), 291 (382), 392 (45), 547 (109)
   in 0.1 N HCl-MeOH (1:9): 260 (237), 291 (382), 391 (45), 546 (115)
   in 0.1N NaOH-MeOH (1:9): 292 (238), 457 (117), 482 (130), 679 (61)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1625, 1455, 1410, 1120, 1055 (as shown in Figure 4)
FAB-MS m/z:
   Positive: 1732 (M+H+Na)⁺
   Negative: 1709 (M+2H-H)⁻
Elemental Analysis: C 57.27%; H 6.27%; N <0.3%
HPLC Rt (min.): 9.7
TLC Rf: 0.38
¹H-NMR (CDCl₃): 0.76 (3H, t), 0.83 (3H, t), 0.93 (3H, d), 0.98 (1H, m), 1.04 (3H, d), 1.07 (1H, m), 1.19 (3H, d), 1.25-1.38 (5H), 1.33 (3H, d), 1.36 (3H, d), 1.36 (3H, d), 1.38-2.08 (18H), 1.91 (1H), 1.97 (1H), 2.02 (1H), 2.15-2.38 (4H), 2.24 (3H, s), 2.38 (3H, s), 2.43 (1H, dd), 2.46 (1H, dd), 2.49 (1H, d), 2.53 (1H, d), 2.79 (1H, qui), 2.95 (1H, d), 3.11-3.24 (3H), 3.24 (1H), 3.30 (1H), 3.52 (1H), 3.52 (1H), 3.54 (1H), 3.62 (1H, dt), 3.75 (1H, d), 3.76 (1H), 3.78-3.90 (3H), 3.98 (1H, d), 3.99 (1H), 4.02 (1H), 4.05 (1H), 4.10-4.22 (3H), 4.12 (3H, s), 4.18 (3H, s), 4.22-4.33 (3H), 4.34 (1H), 4.49 (1H, dd), 4.53 (1H, dd), 4.62 (1H, dd), 4.66 (1H, s), 4.69 (1H, s), 4.93 (1H, d), 5.09 (1H, d), 5.47 (2H, t), 5.86 (1H, s), 7.59 (1H, s), 12.61 (1H, s), 13.27 (1H, s), 14.05 (1H, s) (as shown in Figure 10)
BU-4803T C₂
Nature: purple powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide and methylene chloride
   Slightly soluble in: Methanol and acetonitrile
   Insoluble in: Acetone, hexane and water
UV λ_max nm (E

):
   in MeOH: 261 (257), 292 (392), 394 (47), 552 (102)
   in 0.1 N HCl-MeOH (1:9): 260 (279), 291 (427), 392 (51), 548 (123)
   in 0.1N NaOH-MeOH (1:9): 292 (302), 457 (139), 482 (151), 676 (74)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1695, 1625, 1455, 1410, 1125, 1055 (as shown in Figure 5)
FAB-MS m/z:
   Positive: 1674 (M+H+Na)⁺
   Negative: 1651 (M+2H-H)⁻
HPLC Rt (min.): 10.8
TLC Rf: 0.30
¹H-NMR (CDCl₃): 0.76 (3H, t), 0.84 (3H, t), 0.93 (3H, d), 0.99 (1H, m), 1.09 (1H, m), 1.17 (3H, d), 1.25-1.38 (5H), 1.33 (3H, s), 1.33 (3H, s), 1.34 (3H, s), 1.42-2.08 (17H), 1.92 (1H), 2.15-2.30 (4H), 2.24 (3H, s), 2.41 (1H, dd), 2.46 (1H, dd), 2.49 (1H, d); 2.52 (1H, d), 2.62 (6H, s), 2.78 (1H, qui), 2.88 (1H, d), 3.09-3.30 (6H), 3.49 (1H, dt), 3.52 (1H), 3.53 (1H, dt), 3.75 (1H, d), 3.78-3.86 (3H), 3.95 (1H, d), 4.01 (1H, d), 4.04 (1H, d), 4.10-4.20 (3H), 4.11 (3H, s), 4.17 (3H, s), 4.24-4.30 (3H), 4.34 (1H), 4.43 (1H, dd), 4.50 (1H, d), 4.53 (1H), 4.66 (1H), 4.84 (1H), 4.93 (1H, d), 5.42 (1H, d), 5.46 (1H, d), 5.86 (1H, s), 7.58 (1H, s), 12.63 (1H, s), 13.36 (1H, s), 14.09 (1H, s) (as shown in Figure 11)
BU-4803T D
Nature: purple powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide and methylene chloride
   Slightly soluble in: Methanol and acetonitrile
   Insoluble in: Acetone, hexane and water
UV λ_max nm (E

):
   in MeOH: 261 (217), 291 (347), 395 (39), 548 (97)
   in 0.1 N HCl-MeOH (1:9): 260 (243), 291 (383), 392 (45), 545 (113)
   in 0.1N NaOH-MeOH (1:9): 292 (234), 457 (113), 482 (126), 677 (60)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1625, 1455, 1410, 1125, 1055 (as shown in Figure 6)
FAB-MS m/z:
   Positive: 1717 (M+2H+Na)⁺
   Negative: 1693 (M+2H-H)⁻
Elemental Analysis: C 58.32%; H 6.34%; N <0.3%
HPLC Rt (min.): 17.0
TLC Rf: 0.25
¹H-NMR (CDCl₃): 0.77 (3H, t), 0.84 (3H, t), 0.93 (3H, d), 0.94 (3H, d), 0.99 (1H), 1.07 (1H, m), 1.17 (3H, d), 1.24-1.36 (4H), 1.33 (3H, d), 1.33 (3H, d), 1.34 (3H, d), 1.45-1.86 (6H), 1.88-2.06 (5H), 1.92 (1H), 2.19 (2H), 2.24 (2H), 2.24 (3H, s), 2.24 (3H, s), 2.42 (1H, dd), 2.46 (1H, dd), 2.46 (1H, d), 2.51 (1H, d), 2.78 (1H, qui), 2.88 (1H, brs), 3.11 (1H), 3.13 (1H), 3.18 (1H, dd), 3.25 (2H, m), 3.45 (1H, d), 3.51 (2H), 3.52 (1H, dd), 3.54 (1H, dd), 3.75 (1H, d), 3.76 (1H), 3.78-3.90 (3H), 3.97 (1H, d), 4.01 (1H, d), 4.04 (1H, d), 4.10-4.20 (2H), 4.11 (3H, s), 4.17 (3H, s), 4.24-4.30 (4H), 4.35 (1H), 4.47 (1H, dd), 4.53 (1H, dd), 4.66 (1H), 4.81 (1H), 4.93 (1H, d), 4.93 (1H, d), 5.43 (1H, d), 5.47 (1H, d), 5.86 (1H, s), 7.58 (1H, s), 12.62 (1H, s), 13.37 (1H, s), 14.11 (1H, s)
¹³C-NMR (CDCl₃): 210.5 s, 210.5 s, 203.7 s, 195.3 s, 183.2 s, 176.1 s, 161.2 s, 158.6 s, 152.2 s, 152.1 s, 151.6 s, 150.2 s, 147.6 s, 145.8 s, 143.2 s, 136.4 s, 125.5 s, 125.3 s, 116.3 s, 116.2 s, 113.7 s, 112.6 d, 112.8 s, 107.5 s, 103.3 d, 103.3 d, 99.0 d, 99.0 d, 98.9 d, 98.4 d, 86.8 d, 86.0 s, 85.7 d, 82.9 s, 79.4 s, 78.9 d, 78.8 d, 78.6 s, 77.2 s, 77.2 s, 76.7 d, 75.8 d, 75.4 d, 75.2 d, 75.0 d, 72.9 d, 72.7 d, 70.8 d, 67.9 d, 67.4 d, 67.4 d, 66.7 d, 66.7 d, 65.4 d, 65.1 d, 62.2 q, 61.6 q, 55.4 d, 44.0 d, 36.7 t, 35.3 t, 34.9 t, 34.1 t, 30.5 t, 30.4 t, 29.5 t, 29.4 t, 28.5 t, 27.8 t, 27.8 t, 25.0 q, 25.0 q, 24.9 t, 24.9 t, 18.1 q, 18.1 q, 18.1 q, 17.8 q, 17.7 t, 16.6 t, 15.1 q, 14.8 q, 14.6 q, 14.6 q

[0018] BU-4803T A₁, A₂ and B are produced by fermentation of Microbispora sp. strain AA9966 (ATCC 55327); BU-4803T C₁, C₂ and D are prepared from BU-4803T A₁, A₂ and B, respectively.

Producing Organism

[0019] An actinomycete strain AA9966 which produces new antitumor antibiotics, BU-4803T A₁, A₂ and B, has been isolated from a soil sample collected at Kitakoma-gunn, Yamanashi prefecture, Japan. A biologically pure culture of strain AA9966 identified as Microbispora sp. has been deposited with the American Type Culture Collection (ATCC) under the BUDAPEST TREATY ON THE INTERNATIONAL RECOGNITION OF THE DEPOSIT OF MICROORGANISMS FOR THE PURPOSES OF PATENT PROCEDURE, and all restrictions on the availability to the public of the deposited microorganism will be irrevocably removed upon the granting of a patent from this application. The deposited culture has been assigned the accession number ATCC S5327.

1. Morphology.

[0020] Strain AA9966 is cultivated on oatmeal agar (ISP No. 3) at 28 °C for 21 days. Under light microscope, it is observed that characteristic longitudinal pair spores are formed on aerial mycelium, but not on substrate mycelium. Under scanning electron microscope, it is observed that the spores are oval in shape, 1.5 x 1.2 µm in size, and have smooth surface.

2. Cultural and Physiological Characteristics.

[0021] Cultural and physiological characteristics are determined by cultivating strain AA9966 at 28 °C for 14 to 21 days on various media described by: 1) E.B. Shirling and D. Gottlieb in "Methods for Characterization of Streptomyces species," Intern. J. Syst. Bact. 16:313-340, 1966; 2) S.A. Waksman in The Actinomycetes. Vol. II. Classification, Identification and Description of Genera and Species, pp. 328-334, The Williams & Wilkins Co., Baltimore, 1961; 3) T. Arai in Culture Media for Actinomycetes (The Society for Actinomycetes Japan, 1975); and 4) G.F. Gauze, T.P. Preobrazhenskaya, E.S. Kudrina, N.O. Blinov, I.D. Ryabova and M.A. Sveshnikova in Problems in the Classification of Antagonistic Actinomycetes. State Publishing House for Medical Literature (in Russian), Medzig, Moscow, 1957. Color of mycelia and soluble pigments are assigned using the Manual of Color Names (Japan Color Enterprise Co., Ltd., 1987). Temperature range for growth is determined on yeast starch agar using a temperature gradient incubator TN-3 (Toyo Kagaku Sangyo Co., Ltd.).

[0022] Strain AA9966 grows relatively well on various media. The aerial mycelium is visible on all media except for glycerol nitrate agar, ISP-4 and -7 and nutrient agar. Table 1 shows the results obtained after Strain AA9966 has been cultivated at 28 °C for 21 days.

[0023] Aerial mass color is white to pale yellowish pink. Vegetative mycelium and reverse side of colony are yellowish white to dark yellowish brown. Light grayish blue soluble pigment is observed in oatmeal agar (ISP-3) and oatmeal-yeast extract agar.

[0024] The physiological characteristics of, and the utilization of carbon sources by, strain AA9966 are shown in Tables 2 and 3, respectively.

3. Chemotaxonomy.

[0025] Cell wall composition is analyzed by the method of H.A. Lechevalier and M.P. Lechevalier in "A Critical evaluation of the genera of aerobic actinomycetes," The Actinomycetales (H. Prauser, ed.), pp. 393-405. Jena, Gustav Fischer Verlag, 1970., using thin layer chromatography sheets as described by J.L. Staneck and G.D. Roberts in "Simplified Approach to Identification of Aerobic Actinomycetes by Thin-layer Chromatography," Appl. Microbiol. 28:226-231, 1974. Phospholipid and mycolate composition are determined by the methods of M.P. Lechevalier, C. Debievre and H. Lechevalier in "Chemotaxonomy of Aerobic Actinomycetes: Phospholipid Composition," Biochem. Syst. Ecol. 5: 249-260, 1977 and D.E. Minnikin, L. Alshamaony and M. Goodfellow in "Differentiation of Mycobacterium, Nocardia and Related Taxa by Thin-layer Chromatographic Analysis of Whole-organism Methanolysates," J. Gen. Microbiol. 88: 200-204, 1975, respectively. Menaquinone is analyzed by the procedure of M.D. Collins, H.N. Shah, and D.E. Minnikin in "A Note on the Separation of Natural Mixtures of Bacterial Menaquinones Using Reverse-phase Thin-layer Chromatography," J. Appl. Bacteriol. 48: 277-282, 1980. Fatty acid is determined by the method of K. Suzuki and K. Komagata in "Taxonomic Significance of Cellular Fatty Acid Composition in Some Coryneform Bacteria," Int. J. Syst. Bacteriol. 33: 188-200, 1983.

[0026] Whole cell hydrolysates contain meso- diaminopimelic acid, galactose, glucose, mannose and madurose. Therefore, cell wall type is IIIB. Major phospholipoids include diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol and unknown glucosamine containing-phospholipids. But phosphatidylglycerol, phosphatidylcholine and phosphatidylmethylethanolamine are not detected. Therefore, phospholipid type is PIV type.

[0027] Mycolic acids are not detected. Analysis of the menaquinone composition reveal 47% MK-9(H₄), 22% MK-9(H₂), 13% MK-9(H₆), 11% MK-9(H₈), 5% MK-9(H₀), and 2% MK-9(H₁₀). Fatty acid composition is shown in Table 4.

[0028] The morphological and chemotaxonomic characteristics are consistent with the assignment of strain AA9966 to the genus Microbispora Nonomura and Ohara 1957. Thus, strain AA9966 is identified as Microbispora sp. AA9966.

Table 4. Fatty acid composition of strain AA9966
Fatty acid composition (%)
Straight chain
14:0	15:0	16:0	17:0	18:0
1	5	11	8	2

Branched chain
i-15	i-16	i-17	i-18	a-15	a-17
3	25	2	1	1	8

Unsaturated chain
16:1⁹	i-18:1⁹	10Me16	10Me17	10Me18	i-20H16	20H16
3	1	7	15	3	3	1

Antibiotic Production

[0029] The antitumor antibiotic complex BU-4803T containing the A₁, A₂ and B components is produced by cultivating strain AA9966 or a mutant thereof under submerged conditions in an aqueous nutrient medium. The producing organism is grown in a nutrient medium containing an assimilable carbon source, for example an assimilable carbohydrate. Examples of suitable carbon sources include glucose, cerelose, soluble starch and glycerol. The nutrient medium should also contain an assimilable nitrogen source such as fish meal, yeast extract or ammonium salts. Inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, phosphates, etc. are added if necessary. Trace elements such as copper, manganese, iron, zinc, etc. are added to the medium if desired, or they may be present as impurities of other constituents of the media. The incubation temperature may be any temperature at which the producing strain is able to grow, e.g. from about 20 °C to about 44 °C, but it is preferable to conduct the fermentation at within the range of about 33 °C to about 43 °C. A neutral pH is preferably employed in the medium and production of the antibiotic is generally carried out for a period of about 4 to 8 days. Ordinarily, optimum production is achieved in about 8 days.

[0030] For preparation of relatively small amounts of the antibiotic, shake flask and surface culture can be employed, but for the preparation of larger amounts, submerged aerobic culture in sterile tanks is preferred. When tank fermentation is to be carried out, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating the broth culture with spores from the organism and when a young active vegetative inoculum has been obtained, transferring the inoculum aseptically to the fermentation tank medium. Further agitation may be provided by a mechanical impeller. Antiform agents such as lard oil or silicone oil may also be added if needed. Production of antibiotic BU-4803T complex in the fermentation medium can be readily followed during the course of fermentation by antimicrobial assays using Bacillus subtilis as the test organism.

[0031] It is to be understood that the present invention is not limited to the use of the particular preferred strain AA9966 described above or to organisms fully answering the above descrption. It is especially intended to include other BU-4803T producing strains or mutants of the said organism which can be produced by conventional means such as x-ray radiation, ultraviolet radiation, treatment with nitrogen mustards, phage exposure and the like.

Isolation of BU-4803T A₁, A₂and B

[0032] Antibiotics BU-4803T A₁, A₂ and B may be recovered from the fermentation broth by conventional separation methodologies such as extraction, crystallization and various chromatographic techniques. The following isolation and purification sequence is illustrative.

[0033] The fermentation broth is first extracted with a suitable organic solvent such as ethyl acetate. After the solvent is evaporated, the residue is dissolved in methanol and then chromatographed on an adsorption column such as Diaion HP-20 using aqueous acetone as the eluant to provide a crude BU-4803T complex. The complex is chromatographed on a reversed phase silica gel column to provide a mixture of the A₁ and A₂ components and the B component. The A₁ and A₂ components may be isolated from the mixture by preparative thin layer chromatography using aqueous methanol as the mobile phase. The isolated A₁, A₂ and B components may be further purufied by Sephadex LH-20 column chromatography.

Preparation of BU-4803T C₁, C₂and D.

[0034] BU-4803T C₁, C₂ and D can be prepared from BU-4803T A₁, A₂ and B, respectively. BU-4803T A₁, A₂ and B are stable for at least one month when stored as solid form but are labile when dissolved in dimethyl sulfoxide or methanol or aqueous acetonitrile. Thus, BU-4803T A₁ in dimethyl sulfoxide is converted to BU-4803T C₁ within two hours at room temperature, and within 24 hours when it is dissolved in methanol or aqueous acetonitrile. Similarly, BU-4803T A₂ and B are converted to BU-4803T C₂ and D, respectively, when they are stored in the aforementioned organic solvents.

Biological Properties

[0035] In vitro antimicrobial activities of BU-4803T A₁, A₂, B, C₁, C₂ and D are determined by the serial two-fold agar dilution method using nutrient agar (pH 7.0) and a 5-µl suspension containing 10⁵ cells per ml as inoculum of the test organisms. The minimum inhibitory concentrations (MIC) are determined after incubation for 18 hours at 32 °C. BU-4803T A₁, A₂ and B show inhibitory activity against Gram-positive bacteria with MICs of 0.8 to 12.5 µg/ml, while BU-4803T C₁, C₂ and D do not show inhibitory activity up to 100 µg/ml. The test data are summarized in Table 5.

[0036] BU-4803T A₂, B, C₁, C₂ and D are also tested for their in vitro cytotoxicity against B16-F10 (murine melanoma) and HCT-116 (human colon carcinoma) cells. B16-F10 cells are grown in Eagle's minimum essential medium (Nissui) supplemented with fetal calf serum (FCS, 10%) and kanamycin (60 µg/ml), and HCT-116 cells are grown in McCoy's 5A Medium (Gibco) supplemented with FCS (10%), benzylpenicillin (100 U/ml) and streptomycin (100 µg/ml) at 37 °C under humidified atmosphere in a CO₂ incubator. The above exponentially growing cells are harvested, counted and suspended in the culture media at 1.5 x 10⁴ (B16-F10) and 3.0 x 10⁴ (HCT-116) cells/ml. The cells are planted into wells of a 96-well tissue culture plate and the test compounds are added thereto. The plates are incubated for 72 hours at 37 °C and then the cytotoxic activities are colorimetrically determined at 540 nm after staining viable cells with neutral red solution. All components of BU-4803T showed potent cytotoxicity as shown in Table 6.

Table 6. In vitro cytotoxicity
	IC₅₀ (µg/ml)
	B16-F10	HCT-116
BU-4803T A₁	2.4	4.1
BU-4803T A₂	0.8	2.7
BU-4803T B	0.7	1.9
BU-4803T C₁	1.2	2.5
BU-4803T C₂	3.1	5.3
BU-4803T D	2.7	5.5

[0037] The in vivo antitumor activity of BU-4803T components are determined in tumor-bearing mice. CDF₁ mice are implanted intraperitoneally with 10⁶ cells of P388 leukemia cells per mouse. The test compounds are administered intraperitoneally to the mice once daily on days 1 to 3 (Q1D x 3). At the end of the experiment the median survival time (MST) is determined and used to calculate the %T/C, which is the ratio of the MST of a treated group and the MST of the control group multiplied by 100. A %T/C value of 125 or greater indicates significant antitumor activity. As shown in Table 7, BU-4803T B, C mixture and D show antitumor activity against P388 leukemia in mice.

Table 7. In vivo antitumor activity against P388 leukemia.
Dose (mg/kg/day)	T/C (%)
	30	10	3	1
BU-4803T A mixture		114	123	105
BU-4803T B	109	105	127
BU-4803T C mixture	127	127	109
BU-4803T D		127	109	100

[0038] The invention includes within its scope pharmaceutical compositions containing an effective antimicrobial or tumor-inhibiting amount of an antibiotic of the present invention in combination with an inert pharmaceutically acceptable carrier or diluent. Such compositions may also contain other active antimicrobial or antitumor agents and may be made up in any pharmaceutical form appropriate for the desired route of administration. Examples of such compositions include solid compositions for oral administration such as tablets, capsules, pills, powders and granules, liquid compositions for oral administration such as solutions, suspensions, syrups or elixirs and preparations for parenteral administration such as sterile solutions, suspensions or emulsions. They may also be manufactured in the form of sterile solid compositions which can be dissolved in sterile water, physiological saline or some other sterile injectable medium immediately before use.

[0039] For use as an antitumor agent, optimal dosages and regimens for a given mammalian host can be readily ascertained by those skilled in the art. For use as an antimicrobial agent, an antibiotic of the present invention is administered so that the concentration of the active ingredient is greater than the minimum inhibitory concentration for the particular organism being treated. It will, of course, be appreciated that the actual dose used will vary according to the particular composition formulated, the route of administration and the particular situs, host and disease being treated. Many factors that modify the action of the drug will be taken into account including age, weight, sex, diet, time of administration, route of administration, rate of excretion, condition of the patient, drug combinations, reaction sensitivities and severity of the disease.

[0040] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any manner.

Example 1 Fermentation of strain AA9966

[0041] 

(a) Seed culture. A loopful of mature slant culture of Microbispora sp. AA9966 was inoculated into a 500-ml Erlenmeyer flask containing 100 ml of the seed medium.consisting of soluble starch (Nichiden Kogaku Co.) 2%, glucose 0.5%, NZ-case (Humco Scheffield Chemical Co.) 0.3%, yeast extract (Oriental Yeast Co.) 0.2%,fish extract D30X (Banyu Nutrient Co.) 0.5%, and CaCO₃ 0.3% (pH 7.0). The seed culture was incubated at 28 °C for four days on a rotary shaker (200 rpm).

(b) Production culture. Aliquot (5 ml) of the seed culture was transferred into a 500-ml Erlenmeyer flask containing 100 ml of the production medium consisting of glucose 0.5%, lactose 2%, soluble starch 2%, cornsteep liquor (oji Corn Starch Co.) 1%, Pharmamedia (Trader's Protein) 1.5%, Ebios (dried yeast, Asahi Brewery Co.) 0.2%, and CaCO₃ 0.3% (pH 7.0).

Fermentation was carried out for eight days under the same conditions described above for the seed culture. Antibiotic production in the fermentation broth was monitored by paper disc diffusion assay using Bacillus subtilis PCI219 as the test organism on nutrient agar (pH 6.0). The assay sample was prepared by extracting a 3-ml portion of the whole broth with the same volume of n-BuOH at pH 7.2;the mixture was centrifuged at 3000 rpm for 15 min. and the solvent layer was used in the assay. Antibiotic production reached the maximum potency (ca. 100 µg/ml) after eight days of fermentation.

Example 2 Isolation of BU-4803T A₁, A₂ and B

[0042] The harvested fermentation broth (5 liters) was extracted with ethyl acetate (5 liters) and the extract was concentrated in vacuo to dryness (3.3 g). The extract (1.3 g) was dissolved in methanol (30 ml) and charged on a column of Diaion HP-20 (40 mm i.d. x 150 mm, Mitsubishi Chemical Industries Ltd.). The resin was washed with 50% aqueous methanol (0.5 liter) and then eluted with 80% aqueous acetone (0.5 liter). Evaporation of the eluate yielded a crude BU-4803T mixture (543 mg).

[0043] The BU-4803T mixture (500 mg) was applied on a column of YMC GEL ODS (40 mm i.d. x 450 mm, ODS-AM 120-S50, YMC Co., Ltd), and developed with a mixture of methanol-0.15% KH₂PO₄ adjusted to pH 3.5 (70:30). The eluate was fractionated and the fractions were examined by TLC and HPLC (HPLC conditions: Column: Cosmosil 5C18 AR (4.6 mm i.d. x 150 mm, Nacalai Tesque, Inc.); mobile phase: CH₃CN-H₂O (43:57); flow rate: 1.2 ml/min.; detection: UV absorption at 254 nm. TLC conditions: plate: TLC plate RP-18 F_254S (Merck, Art 15423); solvent: MeOH-H₂O (87.5:12.5). The BU-4803T A mixture was eluted first followed by BU-4803T B. The appropriate fractions of the eluate were pooled and concentrated to aqueous solution. Each solution was extracted with ethyl acetate and the extract was evaporated in vacuo to give BU-4803T A mixture (159.9 mg) and semi-pure BU-4803T B (35.6 mg).

[0044] The semi-pure BU-4803T B was re-chromatographed on a column of Sephadex LH-20 (22 mm i.d. x 450 mm, Pharmacia Fine Chemicals AB) eluting with CH₂Cl₂-methanol (1:1) to give pure BU-4803T B (31.8 mg). BU-4803 T A₁ and A₂ were separated from the A mixture (36 mg) by preparative TLC (RP-18 F_254S, Art. 15389, E. Merck, Darmstadt) using methanol-water (87.5:12.5) as the developing solvent. Two red bands (BU-4803T A₁, Rf 0.49; BU-4803T A₂, Rf 0.43) were scraped off, and each was extracted with CH₂Cl₂ methanol (1:1). Each extract was concentrated and charged on a column of Sephadex LH-20 (20 mm i.d. x 450 mm) eluting with CH₂Cl₂-methanol (1:1). Evaporation of the eluate gave pure BU-4803T A₁ (17.7 mg) and BU-4803T A₂ (5.1 mg).

Example 3 Preparation of BU-4803T C₁, C₂ and D

[0045] BU-4803T B (20 mg) was dissolved in dimethyl sulfoxide (1 ml) and the solution was kept for 15 hours at 4 °C. The solution was charged on a column of YMC D-ODS-5-ST (20 mm i.d. x 150 mm, YMC Co., Ltd.) developed with CH₃CH-H₂O (52:48) at flow rate of 10 ml/min. The eluate was monitored by UV absorption at 300 nm, and the appropriate fractions of the eluate (Rt 17 to 20 min.) were pooled and concentrated to dryness. The residual solid was chromatographed on a column of Sephadex LH-20 (20 mm i.d. x 450 mm) eluting with CH₂Cl₂-methanol (1:1). Evaporation of the eluate gave pure BU-4803T D (10.1 mg).

[0046] Similarly, BU-4803T C₁ (9.1 mg) and C₂ (3.6 mg) were obtained from BU-4803T A₁ (13 mg) and A₂ (5 mg), respectively.

Claims

1. An antibiotic designated BU-4803T A₁ having the following physico-chemical properties:
Nature: red powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide, methanol, acetonitrile, acetone, and methylene chloride
   Insoluble in: Hexane and water
UV λ_max nm (E

):
   in MeOH: 244 (222), 278 (267), 433 (92), 509 (58)
   in 0.1 N HCl-MeOH (1:9): 239 (277), 281 (291), 433 (78), 510 (65)
   in 0.1N NaOH-MeOH (1:9): 236 (217), 278 (292), 437 (119), 613 (51), 648 (50)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1620, 1455, 1405, 1120, 1055 (as shown in Figure 1).
FAB-MS m/z:
   Positive: 1764 (M+H+Na)⁺
   Negative: 1741 (M+2H-H)⁻
Elemental Analysis: C 58.20%; H 6.70%; N <0.3%
HPLC Rt (min.): 10.4
TLC Rf: 0.49
¹H-NMR (CDCl₃): 0.79 (3H, t), 0.87 (3H, t), 0.95 (3H, d), 1.00 (1H, m), 1.04 (3H, d), 1.10 (1H, m), 1.15 (3H, d), 1.29 (3H, d), 1.30 (1H, m), 1.33 (3H, d), 1.34 (2H, m), 1.35 (3H, d), 1.41-1.94 (13H), 1.93 (1H, dd), 1.94 (1H), 1.97 (1H, dd), 2.04 (1H, dt), 2.22 (1H, dt), 2.24-2.38 (2H), 2.24 (3H, s), 2.38 (3H, s), 2.39 (1H, dd), 2.43 (1H, dd), 2.44 (1H), 2.47 (1H, d), 2.64 (1H, d), 2.69 (1H, qui), 3.03 (1H, dd), 3.12 (1H, dt), 3.16 (1H, dt), 3.24-3.33 (2H, dt), 3.34 (1H, d), 3.49 (1H), 3.50-3.60 (2H, dt), 3.64 (1H, dt), 3.68 (1H, dd), 3.72 (1H), 3.73-3.76 (2H), 3.74 (1H), 3.76 (1H), 3.79-3.82 (1H), 3.87 (3H, s), 3.91 (1H, s), 3.95 (3H, s), 3.97 (3H, s), 3.98 (1H), 4.01 (2H, dd), 4.06-4.13 (2H), 4.16 (2H, dd), 4.21 (1H), 4.27 (1H, dd), 4.36 (1H), 4.48 (1H, dd), 4.52 (1H, dd), 4.62 (1H, dd), 4.66 (1H, s), 4.68 (1H, s), 4.94 (1H, d), 5.09 (1H, d), 5.35 (1H, d), 5.43 (1H, d), 5.83 (1H, s), 7.38 (1H, s), 9.69 (1H, s), 12.21 (1H, s), 13.95 (1H, s), 14.96 (1H, s) (as shown in Figure 7)

2. An antibiotic designated BU-4803T A₂ having the following physico-chemical properties:
Nature: red powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide, methanol, acetonitrile, acetone, and methylene chloride
   Insoluble in: Hexane and water
UV λ_max nm (E

):
   in MeOH: 240 (261), 279 (276), 432 (86), 510 (68)
   in 0.1 N HCl-MeOH (1:9): 239 (275), 280 (288), 432 (76), 509 (70)
   in 0.1N NaOH-MeOH (1:9): 235 (214), 278 (279), 436 (113), 615 (54), 647 (54)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1620, 1455, 1405, 1120, 1055 (as shown in Figure 2)
FAB-MS m/z:
   Positive: 1705 (M+Na)⁺
   Negative: 1683 (M+2H-H)⁻
HPLC Rt (min.): 11.1
TLC Rf: 0.43
¹H-NMR (CDCl₃): 0.78 (3H, t), 0.87 (3H, t), 0.95 (3H, d), 0.99 (1H, m), 1.05 (1H, m), 1.14 (3H, d), 1.25-1.34 (5H), 1.29 (3H, d), 1.30 (3H, d), 1.31 (3H, d), 1.44-2.07 (15H), 1.93 (1H), 1.98 (1H), 2.00 (1H), 2.23 (2H), 2.24 (3H, s), 2.26 (1H), 2.39 (1H, dd), 2.43 (1H, dd), 2.44 (1H), 2.47 (1H), 2.63 (1H, d), 2.69 (1H, qui), 3.03 (1H, dd), 3.12 (1H, dt), 3.16 (1H, dt), 3.20-3.30 (3H), 3.36 (1H, d), 3.49 (1H), 3.47-3.55 (2H), 3.68 (1H, dd), 3.72 (1H), 3.74-3.76 (2H), 3.80 (1H), 3.79-3.82 (1H), 3.87 (3H, s), 3.91 (1H, s), 3.93 (1H, s), 3.95 (3H, s), 3.97 (3H, s), 3.99 (1H), 4.02 (3H), 4.02 (1H), 4.06-4.10 (1H), 4.13-4.17 (2H), 4.21 (1H), 4.27 (1H), 4.35 (1H), 4.42 (1H), 4.49 (1H, dd), 4.52 (1H, d), 4.66 (1H), 4.82 (1H), 4.93 (1H, d), 5.34 (1H, d), 5.43 (1H, d), 5.83 (1H, s), 7.38 (1H, s), 9.69 (1H, s), 12.20 (1H, s), 13.94 (1H, s), 14.96 (1H, s) (as shown in Figure 8)

3. An antibiotic herein designated BU-4803T B having the following physico-chemical properties:
Nature: red powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide, methanol, acetonitrile, acetone, methylene chloride
   Insoluble in: Hexane and water
UV λ_max nm (E

):
   in MeOH: 240 (239), 278 (254), 432 (80), 511 (62)
   in 0.1 N HCl-MeOH (1:9): 239 (260), 281 (271), 431 (71), 510 (67)
   in 0.1N NaOH-MeOH (1:9): 235 (211), 278 (272), 436 (111), 614 (51), 647 (51)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1705, 1620, 1460, 1405, 1125, 1060 (as shown in Figure 3)
FAB-MS m/z:
   Positive: 1749 (M+2H+Na)⁺
   Negative: 1725 (M+2H-H)⁻
Elemental Analysis: C 58.45%; H 6.48%; N <0;3%
HPLC Rt (min.): 18.0
TLC Rf: 0.39
¹H-NMR (CDCl₃): 0.77 (3H, t), 0.85 (3H, t), 0.92 (3H, d), 0.93 (3H, d), 0.97 (1H, m), 1.06 (1H, dt), 1.12 (3H, d), 1.27 (3H, d), 1.30 (1H), 1.31 (3H, d), 1.31 (3H, d), 1.32 (2H), 1.45-1.66 (2H), 1.48 (2H), 1.62 (2H), 1.62-1.96 (4H), 1.66 (2H), 1.88 (1H, dt), 1.92 (1H, dd), 1.93 (1H, dd), 1.98 (2H, m), 1.98 (1H, s), 2.18 (2H), 2.20 (2H), 2.22 (3H, s), 2.23 (3H, s), 2.38 (1H, dd), 2.41 (1H, dd), 2.49 (1H), 2.52 (1H), 2.63 (1H, d), 2.67 (1H, qui), 3.02 (1H, dd), 3.10 (1H, dd), 3.13 (1H, dd), 3.24 (2H, dt), 3.37 (1H, d), 3.47-3.56 (2H), 3.54 (1H, s), 3.55 (1H, s), 3.66 (1H, m), 3.73 (1H, m), 3.74 (1H), 3.80 (1H, m), 3.85 (3H, s), 3.93 (3H, s), 3.95 (1H), 3.95 (3H, s), 3.96 (1H, d), 4.00 (1H, d), 4.08-4.23 (3H, m), 4.25 (1H, dt), 4.25 (1H, dt), 4.37 (1H, s), 4.45 (1H, d), 4.51 (1H, d), 4.66 (1H, s), 4.80 (1H, s), 4.92 (1H, d), 4.92 (1H, d), 5.32 (1H, d), 5.40 (1H, d), 5.81 (1H, s), 7.37 (1H, s), 9.69 (1H, s), 12.20 (1H, s), 13.94 (1H, s), 14.96 (1H, s) (as shown in figure 9)
¹³C-NMR (CDCl₃): 210.6 s, 210.6 s, 203.5 s, 195.5 s, 187.8 s, 184.8 s, 163.6 s, 158.3 s, 156.8 s, 153.2 s, 151.8 s, 150.6 s, 147.7 s, 139.4 s, 138.5 s, 135.4 s, 125.6 s, 124.7 s, 116.3 s, 113.0 s, 112.1 d, 110.5 s, 108.2 s, 107.9 s, 103.2 d, 103.2 d, 98.9 d, 98.9 d, 98.9 d, 98.7 d, 86.6 d, 85.6 s, 85.4 d, 82.7 s, 79.3 s, 78.9 d, 78.8 d, 78.5 s, 77.2 s, 77.2 s, 77.0 d, 76.3 d, 75.4 d, 75.3 d, 75.2 d, 72.7 d, 72.5 d, 70.6 d, 67.8 d, 67.1 d, 67.0 d, 66.7 d, 66.7 d, 65.1 d, 65.0 d, 61.1 q, 60.9 q, 60.8 q, 55.7 d, 44.4 d, 37.0 t, 35.3 t, 34.8 t, 34.3 t, 30.5 t, 30.4 t, 29.5 t, 29.4 t, 27.8 t, 27.8 t, 27.8 t, 25.0 q, 25.0 q, 24.8 t, 24.8 t, 18.1 q, 18.1 q, 18.0 t, 17.9 q, 17.7 q, 16.6 t, 15.1 q, 14.9 q, 14.6 q, 14.6 q

4. An antibiotic designated as BU-4803T C₁ having the following physico-chemical properties:
Nature: purple powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide and methylene chloride
   Slightly soluble in: Methanol and acetonitrile
   Insoluble in: Acetone, hexane and water
UV λ_max nm (E

):
   in MeOH: 260 (237), 291 (382), 392 (45), 547 (109)
   in 0.1 N HCl-MeOH (1:9): 260 (237), 291 (382), 391 (45), 546 (115)
   in 0.1N NaOH-MeOH (1:9): 292 (238), 457 (117), 482 (130), 679 (61)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1625, 1455, 1410, 1120, 1055 (as shown in Figure 4)
FAB-MS m/z:
   Positive: 1732 (M+H+Na)⁺
   Negative: 1709 (M+2H-H)⁻
Elemental Analysis: C 57.27%; H 6.27%; N <0.3%
HPLC Rt (min.): 9.7
TLC Rf: 0.38
¹H-NMR (CDCl₃): 0.76 (3H, t), 0.83 (3H, t), 0.93 (3H, d), 0.98 (1H, m), 1.04 (3H, d), 1.07 (1H, m), 1.19 (3H, d), 1.25-1.38 (5H), 1.33 (3H, d), 1.36 (3H, d), 1.36 (3H, d), 1.38-2.08 (18H), 1.91 (1H), 1.97 (1H), 2.02 (1H), 2.15-2.38 (4H), 2.24 (3H, s), 2.38 (3H, s), 2.43 (1H, dd), 2.46 (1H, dd), 2.49 (1H, d), 2.53 (1H, d), 2.79 (1H, qui), 2.95 (1H, d), 3.11-3.24 (3H), 3.24 (1H), 3.30 (1H), 3.52 (1H), 3.52 (1H), 3.54 (1H), 3.62 (1H, dt), 3.75 (1H, d), 3.76 (1H), 3.78-3.90 (3H), 3.98 (1H, d), 3.99 (1H), 4.02 (1H), 4.05 (1H), 4.10-4.22 (3H), 4.12 (3H, s), 4.18 (3H, s), 4.22-4.33 (3H), 4.34 (1H), 4.49 (1H, dd), 4.53 (1H, dd), 4.62 (1H, dd), 4.66 (1H, s), 4.69 (1H, s), 4.93 (1H, d), 5.09 (1H, d), 5.47 (2H, t), 5.86 (1H, s), 7.59 (1H, s), 12.61 (1H, s), 13.27 (1H, s), 14.05 (1H, s) (as shown in Figure 10)

5. An antibiotic desingated as BU-4803T C₂ having the following physico-chemical properties:
Nature: purple powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide and methylene chloride
   Slightly soluble in: Methanol and acetonitrile
   Insoluble in: Acetone, hexane and water
UV λ_max nm (E

):
   in MeOH: 261 (257), 292 (392), 394 (47), 552 (102)
   in 0.1 N HCl-MeOH (1:9): 260 (279), 291 (427), 392 (51), 548 (123)
   in 0.1N NaOH-MeOH (1:9): 292 (302), 457 (139), 482 (151), 676 (74)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1695, 1625, 1455, 1410, 1125, 1055 (as shown in Figure 5)
FAB-MS m/z:
   Positive: 1674 (M+H+Na)⁺
   Negative: 1651 (M+2H-H)⁻
HPLC Rt (min.): 10.8
TLC Rf: 0.30
¹H-NMR (CDCl₃): 0.76 (3H, t), 0.84 (3H, t), 0.93 (3H, d), 0.99 (1H, m), 1.09 (1H, m), 1.17 (3H, d), 1.25-1.38 (5H), 1.33 (3H, s), 1.33 (3H, s), 1.34 (3H, s), 1.42-2.08 (17H), 1.92 (1H), 2.15-2.30 (4H), 2.24 (3H, s), 2.41 (1H, dd), 2.46 (1H, dd), 2.49 (1H, d), 2.52 (1H, d), 2.62 (6H, s), 2.78 (1H, qui), 2.88 (1H, d), 3.09-3.30 (6H), 3.49 (1H, dt), 3.52 (1H), 3.53 (1H, dt), 3.75 (1H, d), 3.78-3.86 (3H), 3.95 (1H, d), 4.01 (1H, d), 4.04 (1H, d), 4.10-4.20 (3H), 4.11 (3H, s), 4.17 (3H, s), 4.24-4.30 (3H), 4.34 (1H), 4.43 (1H, dd), 4.50 (1H, d), 4.53 (1H), 4.66 (1H), 4.84 (1H), 4.93 (1H, d), 5.42 (1H, d), 5.46 (1H, d), 5.86 (1H, s), 7.58 (1H, s), 12.63 (1H, s), 13.36 (1H, s), 14.09 (1H, s) (as shown in Figure 11)

6. An antibiotic designated as BU-4803T D having the following physico-chemical properties:
Nature: purple powder
M.P. (°C, dec.): >200
Solubility:
   Soluble in: Dimethyl sulfoxide and methylene chloride
   Slightly soluble in: Methanol and acetonitrile
   Insoluble in: Acetone, hexane and water
UV λ_max nm (E

):
   in MeOH: 261 (217), 291 (347), 395 (39), 548 (97)
   in 0.1 N HCl-MeOH (1:9): 260 (243), 291 (383), 392 (45), 545 (113)
   in 0.1N NaOH-MeOH (1:9): 292 (234), 457 (113), 482 (126), 677 (60)
IR ν_max (KBr) cm⁻¹: 3450, 2940, 1700, 1625, 1455, 1410, 1125, 1055 (as shown in Figure 6)
FAB-MS m/z:
   Positive: 1717 (M+2H+Na)⁺
   Negative: 1693 (M+2H-H)⁻
Elemental Analysis: C 58.32%; H 6.34%; N <0.3%
HPLC Rt (min.): 17.0
TLC Rf: 0.25
¹H-NMR (CDCl₃): 0.77 (3H, t), 0.84 (3H, t), 0.93 (3H, d), 0.94 (3H, d), 0.99 (1H), 1.07 (1H, m), 1.17 (3H, d), 1.24-1.36 (4H), 1.33 (3H, d), 1.33 (3H, d), 1.34 (3H, d), 1.45-1.86 (6H), 1.88-2.06 (5H), 1.92 (1H), 2.19 (2H), 2.24 (2H), 2.24 (3H, s), 2.24 (3H, s), 2.42 (1H, dd), 2.46 (1H, dd), 2.46 (1H, d), 2.51 (1H, d), 2.78 (1H, qui), 2.88 (1H, brs), 3.11 (1H), 3.13 (1H), 3.18 (1H, dd), 3.25 (2H, m), 3.45 (1H, d), 3.51 (2H), 3.52 (1H, dd), 3.54 (1H, dd), 3.75 (1H, d), 3.76 (1H), 3.78-3.90 (3H), 3.97 (1H, d), 4.01 (1H, d), 4.04 (1H, d), 4.10-4.20 (2H), 4.11 (3H, s), 4.17 (3H, s), 4.24-4.30 (4H), 4.35 (1H), 4.47 (1H, dd), 4.53 (1H, dd), 4.66 (1H), 4.81 (1H), 4.93 (1H, d), 4.93 (1H, d), 5.43 (1H, d), 5.47 (1H, d), 5.86 (1H, s), 7.58 (1H, s), 12.62 (1H, s), 13.37 (1H, s), 14.11 (1H, s) (as shown in Figure 12)
¹³C-NMR (CDCl₃): 210.5 s, 210.5 s, 203.7 s, 195.3 s, 183.2 s, 176.1 s, 161.2 s, 158.6 s, 152.2 s, 152.1 s, 151.6 s, 150.2 s, 147.6 s, 145.8 s, 143.2 s, 136.4 s, 125.5 s, 125.3 s, 116.3 s, 116.2 s, 113.7 s, 112.6 d, 112.8 s, 107.5 s, 103.3 d, 103.3 d, 99.0 d, 99.0 d, 98.9 d, 98.4 d, 86.8 d, 86.0 s, 85.7 d, 82.9 s, 79.4 s, 78.9 d, 78.8 d, 78.6 s, 77.2 s, 77.2 s, 76.7 d, 75.8 d, 75.4 d, 75.2 d, 75.0 d, 72.9 d, 72.7 d, 70.8 d, 67.9 d, 67.4 d, 67.4 d, 66.7 d, 66.7 d, 65.4 d, 65.1 d, 62.2 q, 61.6 q, 55.4 d, 44.0 d, 36.7 t, 35.3 t, 34.9 t, 34.1 t, 30.5 t, 30.4 t, 29.5 t, 29.4 t, 28.5 t, 27.8 t, 27.8 t, 25.0 q, 25.0 q, 24.9 t, 24.9 t, 18.1 q, 18.1 q, 18.1 q, 17.8 q, 17.7 t, 16.6 t, 15.1 q, 14.8 q, 14.6 q, 14.6 q.

7. A process for the preparation of an antibiotic designated BU-4803T A₁ which comprises cultivating a strain of Microbispora capable of producing said antibiotic in an aqueous medium containing an assimilable source of carbon and nitrogen under aerobic condition, and recovering said antibiotic from the cultured broth.

8. A process for the preparation of an antibiotic designated BU-4803T A₂ which comprises cultivating a strain of Microbispora capable of producing said antibiotic in an aqueous medium containing an assimilable source of carbon and nitrogen under aerobic condition, and recovering said antibiotic from the cultured broth.

9. A process for the preparation of an antibiotic designated BU-4803T B which comprises cultivating a strain of Microbispora capable of producing said antibiotic in an aqueous medium containing an assimilable source of carbon and nitrogen under aerobic condition, and recovering said antibiotic from the cultured broth.

10. A biologically pure culture of the microorganism Microbispora strain AA9966 having the identifying characteristics of ATCC 55327 and is capable of producing an antibiotic selected from the group consisting of BU-4803T A₁, BU-4803T A₂ and BU-4803T B upon cultivation in an aqueous medium containing an assimilable source of carbon and nitrogen.

Drawing